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Step-and-Repeat Photopatterning of Protein Features Using Caged-Biotin-BSA: Characterization and Resolution A. S. Blawas, T. F. Oliver, M. C. Pirrung, and W. M. Reichert* Department of Biomedical Engineering and NSF/ERC Center of Emerging Cardiovascular Technologies, Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708 Received November 12, 1997. In Final Form: May 9, 1998 This paper characterizes the caged-biotin-BSA system developed for selectively patterning biotinylated proteins into patterns on glass slides. Methyl R-nitropiperonyloxycarbonyl biotin, caged biotin, is coupled to a bovine serum albumin (BSA) carrier molecule using succinimide chemistry and then employed in a four-step patterning process: (1) caged-biotin-BSA compound is adsorbed onto a glass slide; (2) the slide is irradiated with 353-nm light through a chrome-on-glass photomask; (3) streptavidin is selectively bound to the irradiated regions; and (4) biotinylated protein is bound to the selectively immobilized streptavidin. A step-and-repeat scheme was used to demonstrate that streptavidin and then biotinylated BSA can be sequentially immobilized with reproducible feature density and little interfeature binding. Eight-minute irradiation of a mixed monolayer of 25% native BSA and 75% caged-biotin-BSA yielded the highest feature contrast, required the minimum use of reagent, and produced the least nonspecific binding. FITClabeled streptavidin and biotinylated BSA served as the patterned protein films. Pattern resolution decreased with both decreasing feature size and increasing substrate thickness, primarily due to pattern spreading effects. The molecular density of the patterned protein, determined via fluorescence microscopy, is 3.9 × 1010 molecules/mm2 for caged-biotin-BSA, 9 × 109 molecules/mm2 for patterned streptavidin, and 1.5 × 109 molecules/mm2 for biotinylated BSA. These results indicate that the immobilization of streptavidin and of biotinylated BSA (steps 3 and 4) are both on the order of 20% efficient, for an overall efficiency of approximately 4%. A series of three step-and-repeat procedures are used to produce a pattern of three different biotinylated and fluorescently labeled proteins.
Introduction Although the field of protein patterning is relatively new, at least three patterning methodologies have emerged: traditional photoresist technology,1,2 self-assembled monolayers,3,4 and photochemistry.5-7 The most frequently cited applications for protein patterning are patterned cell growth8 and patterned antibodies for immunoassay.9 A recent critique of these methodologies and assessment of patterning applications is available.10 For protein patterns to be implemented technologically, they must exhibit uniform density, acceptable resolution, and reproducibility. Many of the recent patterning reports * To whom correspondence should be addressed. Phone: (919) 660-5151. Fax: (919) 684-4488. E-mail:
[email protected]. (1) Britland, S.; Perez-Arnaud, E.; Clark, P.; McGinn, B.; Connolly, P.; Moores, G. Biotechnol. Progr. 1992, 8, 155-160. (2) Healy, K. E.; Thomas, C. H.; Rezania, A.; Kim, J. E.; McKeown, P. J.; Lom, B.; Hockberger, P. E. Biomaterials; 17, pp 195-208 1996. (3) Bhatia, S. K.; Teixeira, J. L.; Anderson, M.; Shriver-Lake, L. C.; Calvert, J. M.; Georger, J. H.; Hickman, J. J.; Dulcey, C. S.; Schoen, P. E.; Ligler, F. S. Anal. Biochem. 1993, 208, 197-205. (4) Kumar, A.; Abbott, N.; Kim, E.; Biebuyck, H.; Whitesides, G. Acc. Chem. Res. 1995, 28, 219-226. (5) Fodor, S.; Leighton Read, J.; Pirrung, M.; Stryer, L.; Tsai Lu, A.; Solas, D. Science 1991, 251, 767-772. (6) Gao, H.; Sa¨nger, M.; Luginbu¨hl, R.; Sigrist, H. Biosens. Bioelectron. 1995, 10, 317-328. (7) Morgan, H.; Pritchard, D. J.; Cooper, J. M. Biosens. Bioelectron. 1995, 10, 841-846. (8) Hockberger, P.; Lom, B.; Soekarno, A.; Healey, K. Nanofabrication and biosystems: integrating material science, engineering and biology. Cellular engineering control of cell-substrate interactions. Hoch, H.; Jelinski, H.; Craighead, H., Eds.; Cambridge University Press: New York, 1996. (9) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 3605-3607. (10) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609.
have achieved acceptable image resolution, perhaps most notably by Lo´pez et al.11 who reported single protein patterns on the order of 2 µm. Two elements noticeably absent from the protein patterning literature are (1) critical assessments of the patterned film uniformity, density, resolution, and reproducibility, and (2) a quantitative demonstration of sequential protein patterns. A worthy paradigm for these criteria comes from integrated circuit (IC) processing technology which must produce tightly packed, clearly resolved, and reproducible patterned features.12,13 There are two primary types of IC patterning: singlepass patterning and step-and-repeat patterning. Singlepass patterning is when an entire pattern is created simultaneously after one series of irradiation, etching, and deposition steps. Step-and-repeat patterning is used to produce multiple features sequentially by a series of processing steps. By analogy, the field of protein patterning has focused almost exclusively on single-pass patterning: a surface is prepared, goes through one round of processingseither irradiation, stamping, or chemical treatmentsand is ready for application. In single-pass patterning, it is important to have uniform protein density across a pattern for consistency of application. The same is true for step-and-repeat protein patterning, except that the deposition of equal protein density must be repeated during each subsequent processing step. (11) Lo´pez, G. P.; Biebuyuck, H.; Kumar, A.; Whitesides, G. J. Am. Chem. Soc. 1993, 115, 10774-10781. (12) Sukanek, P.; Wilcox, W. R.; Ryan, J. G. Integrated circuit manufacture. Encyclopedia of Physical Science and Technology; Academic Press: New York, 1992; Vol. 8, pp 173-195. (13) White, L. K. RCA Rev. 1986, 47, 345-379.
S0743-7463(97)01231-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/24/1998
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Figure 1. Methyl R-nitropiperonyloxycarbonyl biotin, caged biotin, is coupled to a BSA carrier molecule using succinimide chemistry and then employed in a four-step process: (a) the caged-biotin-BSA aggregate is adsorbed to a glass slide; (b) the slide is irradiated with 353-nm light through a chromeon-glass photomask; (c) streptavidin is selectively bound to the irradiated regions; and (d) biotinylated protein is bound to the selectively immobilized streptavidin.
The intended goal of our work is to create multiple protein patterns for single-chip multianalyte immunoassay. The method selected to achieve this is a step-andrepeat photolithographic immobilization of biotinylated antibodies through a patterned streptavidin intermediate. Fodor et al.5 were the first to describe photodeprotection chemistry for the purpose of producing peptide arrays. Sundberg et al.14 subsequently showed that photoprotected caged biotin can be used to selectively immobilize multiple biotinylated proteins on surfaces using a streptavidin intermediate. More recently, Pirrung and Huang15 described the patterning of proteins where the caged biotin was immobilized via the adsorption of a bovine serum albumin (BSA) carrier. The BSA protein carrier was used to facilitate the immobilization of caged biotin and reduce nonspecific binding of protein during the post-irradiation protein immobilization steps.16 The caged-biotin-BSA patterning scheme is illustrated in Figure 1 for a single antibody immobilization. Following (a) preadsorption of glass slide with a layer of cagedbiotin-BSA, the slide is (b) selectively deprotected through a photomask, followed by (c) binding of streptavidin, and then (d) biotinylated protein. Translating the sample with respect to the photomask and repeating the last three steps form a continuous step-and-repeat protocol for producing arrays of proteins. This paper quantitatively reports the protein densities, feature resolution, and feature profile of step-and-repeat patterns of streptavidin and biotinylated BSA. Materials and Methods Caged-Biotin-BSA Layer Preparation. Synthesis of the methyl R-nitropiperonyloxycarbonyl (MeNPOC) caging group and the coupling of MeNPOC to biotin are described elsewhere,15 but with two necessary modifications. First, the methylnitropiperonyl alcohol was coupled to biotin methyl ester with 4-nitrophenyl chloroformate14 instead of with carbonyldiimidazole. The resulting nitrophenyl carbonate was converted to the MeNPOC(14) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. J. Am. Chem. Soc. 1995, 117, 1205012057. (15) Pirrung, M. C.; Huang, C.-Y. Bioconjugate Chem. 1996, 7, 31721. (16) Vogt, R.; Phillips, D.; Henderson, O.; Whitfield, W.; Spierto, F. J. Immmunol. Meth. 1987, 101, 43-50.
Blawas et al. biotin-OMe utilizing sodium hydride as before. Second, the NHS-esters were generated using polymer bound 1-ethyl-3-(3dimethylaminopropyl)carbdiimide (P-EDAC).17,18 Caged-biotin-BSA was generated by adding 5 mg/mL caged biotin succinimide ester solution to a solution of BSA (4 mg) (Sigma, fraction V, Mw 68 000) in 2 mL of a carbonate/bicarbonate buffer. The final coupling ratio was determined by the UV absorption at 353 nm for caged biotin ( ) 4595 M-1) and 280 nm for BSA ( ) 45 000 M-1). The calculation was corrected for the absorbance of MeNPOC at 280 nm ( ) 283 M-1). Caged biotin labeling ratios from 5 to 30 were prepared on the basis of the efficiency of the ester coupling reaction. The caged biotin labeling was optimized through two procedures (unpublished results). Fluorescence Labeling. FITC-labeled streptavidin (Sigma, 8:1 labeling ratio, Mw ∼69 000) was purchased and used without further purification. Biotinylated BSA was purchased (Sigma, 9:1 labeling ratio Mw ∼73 000) and labeled with fluorescein isothiocyanate (FITC, Sigma). Twenty microliters of a 10 mg/ mL solution of FITC (1 mg) in anhydrous dimethylformamide (Aldrich) was added to separate vortexing solutions of BSA (1 mg), caged-biotin-BSA (1 mg), and biotinylated BSA (1 mg) in 1 mL of 0.1 M sodium bicarbonate buffer (pH 9.0). After being incubated for 2 h at rt, unreacted dye was separated from labeled protein by gel filtration on a PD-10 column (Pharmacia). Final labeling ratios of 1:1, 0.6:1, and 1:1 were determined using absorption at 494 nm for FITC ( ) 73 000 M-1) and 280 nm for BSA ( ) 45 000 M-1) for BSA, caged-biotin-BSA and biotinylated BSA, respectively. Goat host biotinylated antibodies (IgG) (Pierce) [antimouse, antihuman, and antirabbit] were purchased and labeled with fluorescein isothiocyanate (FITC, Sigma) in the same manner. Final labeling ratios of 2:1, 3.1:1, and 3.1:1, respectively, were determined using adsorption at 494 nm for FITC ( ) 73 000 M-1) and 280 nm for IgG ( ) 170 000 M-1). Radiolabeling. BSA and BSA-biotin-MeNPOC were labeled with 125I at a level of 1 cpm, using an IODO-GEN (Pierce) procedure. Streptavidin was labeled with 125I at a level of 50 cpm in a similar manner using Bolton-Hunter (Pierce) reagent with a subsequent IODO-GEN (Pierce) reaction. In the radiolabeling experiments, five cleaned glass specimens were adsorbed with a 30 µM (2 mg/mL) solution of 125I-labeled BSA, and five were adsorbed with 125I-labeled 10:1 caged-biotin-BSA. In addition, 10 cleaned glass specimens were preadsorbed with caged-biotin-BSA, five of which were irradiated for 8 min, while the other five specimens were used as unirradiated controls. The irradiated and control specimens were incubated with 3 µM (0.2 mg/mL) solutions of 125I-labeled streptavidin for 15 min. The samples were rinsed three times in PBS (pH 7.4) and placed into labeled tubes for counting on an automated gamma counter (Packard Instruments, Meriden, CT). After 24 h, the samples were removed, dip rinsed, and placed into fresh tubes for counting. The second count was accepted as the final amount of protein adsorbed. Each experimental condition employed five glass samples. Substrate Preparation. One-millimeter-thick soda lime glass slides (Propper Manufacturing Co.) (3 in. × 1 in.) or 0.15mm-thick soda lime glass cover slips (Sigma-Aldrich) (1 in. × 2.5 in.) were cleaned by immersion for 10 min in a 1% solution of H2O2 in concentrated H2SO4 and subsequently washed with copious amounts of DI water. The surface was soaked for 1 h in 95% ethanol and allowed to air-dry. Glass slides were used throughout the study, except for one set of feature resolution measurements. Flow Cell Construction. A flow cell was constructed for the purpose of performing step-and-repeat masked irradiation and protein incubation to sequentially pattern a series of protein features onto a single substrate. The glass substrate fits into a 3.020 in. × 1.015 in. rectangular depression milled into a 3.5in.-diameter, circular piece of black Delrin. The perimeter of the rectangular depression has two concentric grooves that accommodate inner and outer rubber O-rings. The slide is (17) Desai, M. C.; Stephens Stramiello, L. M. Tetrahedron Lett. 1993, 34, 7685. (18) Adamcyzk, M.; Fishpaugh, J. R.; Mattingly, P. Tetrahedron Lett. 1995, 36, 8345-8346.
Step-and-Repeat Protein Patterning positioned on the two gaskets in the rectangular depression. A vacuum is pulled between the two gaskets, creating a watertight seal between the slide and the plastic piece. The flow cell volume consists of the 0.7-mL space between the Delrin piece and the fitted glass slide bounded by the inner rubber gasket. Inlet and outlet ports milled into the plastic piece within the region bounded by the inner gasket are used to introduce reagents to the surface of the glass slide. The flow cell is mounted glass slide up on a stage beneath the irradiation source for precise photomasking of the glass slide. Irradiation Source. The caged-biotin-BSA coated substrate was assembled in a flow cell and placed on a micrometer stage 15 cm below the lamp housing to allow for step-and-repeat processing. The collimated irradiation source was an ORIEL (model 66033, Stratford, CT) 350 W flood source (Hg vapor) ultraviolet lamp with a 3-in.-diameter collimated beam filtered through a 350-360 band-pass filter. The slides were irradiated at an average power of 7.0 ( 0.1 mW/cm2. Photomasking. Two types of photomasks were used: a resolution target and a single feature mask. The resolution target, used to determine the smallest discernible feature size, was a chrome-on-glass USAF negative test target (Melles Griot) consisting of line pairs (a feature and equally wide adjacent space) of decreasing size from 1 mm to 2.19 µm. The single feature mask consisted of a 1-mm slit chrome-on-glass mask (Applied Image, Rochester, NY). Irradiations employed contact masking with the back surface of the derivatized slides. Previous experience with direct contact masking and proximity masking of the derivatized face of the slide yielded unsatisfactory results. Quantitative Pattern Imaging. The patterned slides were rinsed in DI water and allowed to air-dry. Selected regions of the slide (∼10 mm2) were imaged using a BIORAD MRC 1000 confocal microscope (Bio-Rad Microscience Ltd, Hemel Hempstead, U.K.) with a 4× objective and Kalman 10 image average and analyzed using NIH Image 1.59.24 The images consisted of 768 × 512 pixel arrays where one pixel corresponds to 4.6 µm. The confocal images were used to collect quantitative intensities for each patterned feature and the background interfeature regions. A calibration curve for the confocal images was constructed by applying a series of 0.5-µL droplets of streptavidin-FITC and biotin-BSA-FITC solutions (0.6 to 25 µg/mL) to glass slides and allowing them to dry. The average pixel intensity per unit area of each spot, without background subtraction, was correlated with the corresponding protein surface density. Calibration curves were collected at several gain levels so that patterned and background regions could be imaged within the midrange of the instrument (50- to 175-pixel intensity). Whole Pattern Imaging. Overall images of the entire pattern were collected using the waveguide configuration described elsewhere.19 The patterned slide was assembled into the previously described waveguide flow cell and placed on a rotating goniometer. Laser light (488 nm) was coupled into the end of the slide by a hemicylindrical lens. The fluorescence image of the slide was captured by a thermoelectrically cooled CCD camera (Santa Barbara Instruments Group, Inc., Santa Barbara, CA). Images were collected for a period of 20 s and processed using NIH Image 1.59. The image plane of the CCD was 4.6 × 3.0 cm which provides a broad view of the entire patterned region. System Optimization. The caged biotin labeling was optimized through two procedures (unpublished results). First, cleaned glass slides were preadsorbed with BSA coupled with 6, 10, 15, 23, and 28 caged biotins, irradiated through a 1-mm slit photomask, and incubated with FITC-streptavidin. Fluorescence intensities in the irradiated regions were determined using calibrated confocal microscopy. Surfaces adsorbed with BSA coupled with 10 caged biotins (10:1 caged-biotin-BSA) bound the highest average surface density of streptavidin. Second, stock solutions of 10:1 caged-biotin-BSA and unlabeled BSA were mixed to produce protein solutions with ratios of caged-biotinBSA to native BSA of 0:1 (all unlabeled BSA), 1:3, 1:1, 3:1, 1:0 (all caged-biotin-BSA). Cleaned glass slides were preadsorbed (19) Plowman, T. E.; Reichert, W. M.; Peters, C.; Wang, H. K.; Christensen, D. A.; Herron, J. N. Biosens. Bioelectron. 1996, 11, 149160.
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Figure 2. Fraction of streptavidin bound versus irradiation dosage. Ei indicates the irradiation dose required to produce 10% of maximum streptavidin binding (0.5 min). Ef indicates the irradiation dose required to produce 90% of maximum streptavidin binding (7 min). with a mixed monolayer of caged-biotin-BSA and unlabeled BSA, irradiated as above, and then incubated with FITC-streptavidin. Fluorescence intensities in the irradiated and unirradiated regions were compared quantitatively using calibrated confocal microscopy. The relative contrast ratio between patterned features and the unirradiated regions was highest for the 3:1 surface preparation. Combined, these results indicated that irradiation of a 10:1 caged-biotin-BSA mixed in a ratio of 3:1 with unlabeled BSA gave the best results. Protein Patterning Protocol. Unless stated otherwise, the following procedure is employed. Cleaned glass slides were preadsorbed for 1 h with 300 µL of a 30 µM (2 mg/mL) solution of 10:1 caged-biotin-BSA mixed in a ratio of 3:1 with unlabeled BSA and then rinsed in phosphate-buffered saline (pH 7.4). The protein-coated slides were assembled into the flow cell used for protein patterning. All slide manipulation occurred in subdued lighting; however, exposure to room light up to 2 h caused no significant deprotection effects. The slide was irradiated through the photomask for 8 min and then developed by incubating for 15 min with a 1.6 µM (100 µg/mL) streptavidin, followed by a PBS rinse. If a layer of biotinylated BSA was added to the patterned streptavidin, the PBS rinse was followed by a 30-60 min incubation with 1.7 µM (100 µg/mL) solution of FITC-labeled biotinylated BSA (8 biotins per BSA molecule) or FITC-labeled biotinylated IgG (∼7 biotins per IgG molecule). Sequential repetition of the above procedure, minus the caged-biotin-BSA preadsorption, formed the step-and-repeat protein patterning process. Statistical Analysis. Statistical analysis was performed using InStat 2.00 on a MacIntosh PowerPC that employed a Welch’s 2-tailed p test (p < 0.05). Significant differences between mean values and zero were determined using a one-way t test (p < 0.05).
Results Determination of Irradiation Dose. Using the protocols described above, glass slides were preadsorbed with a 3:1 mixture of caged-biotin-BSA and BSA, irradiated through a 1-mm slit photomask, incubated with FITC-streptavidin, and rinsed. Samples were irradiated for periods from 0.5 to 20 min. The fluorescence intensity of the patterned strip was determined by confocal microscopy. Three specimens were examined for each irradiation time. Figure 2 shows normalized fluorescence intensity of bound FITC-streptavidin plotted as a function of irradiation time. Just prior to 10 min is the apparent point of “diminishing returns” beyond which further
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Table 1. Surface Densities of FITC-Labeled Proteins Determined by Calibrated Confocal Microscopy species (n ) 3) 100% BSA 100% caged-biotin-BSA caged-biotin-BSA (3:1 mixture with BSA) streptavidin (pattern) streptavidin (background) biotin-BSA (pattern) biotin-BSA (background) controls: streptavidin on BSA streptavidin on unirradiated BSA-biotin-MeNPOC
ng/mm2
1010 molecules/ mm2
5.4 ( 1.1a 6.8 ( 1.3a 4.4 ( 1.4a
4.7 ( 1.0a 6.0 ( 1.2a 3.9 ( 1.2a
1.0 ( 0.3 0.16 ( 0.09b 0.18 ( 0.03 0.05 ( 0.04b
0.9 ( 0.3 0.14 ( 0.08b 0.16 ( 0.03 0.04 ( 0.03b
0.07 ( 0.004 0.08 ( 0.005
0.06 ( 0.004 0.07 ( 0.005
a Not statistically different from each other (p > 0.05). b Not statistically different from zero (p > 0.05).
irradiation produces little to no increase in the amount of bound protein, presumably owing to negligible increases in the further deprotection of the preadsorbed caged biotin. Ei and Ef, respectively, refer to the irradiation doses necessary to incur 10% and 90% caged biotin photodeprotection (see Discussion section). To maximize the amount of bound protein while minimizing photodegradation from overexposure to irradiation, an irradiation time of 8 min was employed for all subsequent patterning procedures. Protein Surface Densities. Calibrated confocal microscopy and radiolabeling were used to determine the surface densities of BSA adsorbed from a BSA solution and that of caged-biotin-BSA adsorbed from a solutions of 100% caged-biotin-BSA and from a 3:1 mixture of caged-biotin-BSA and BSA. In addition, confocal microscopy was used to determine the surface densities of streptavidin and biotinylated BSA bound to the photomasked specimens preadsorbed with a 3:1 mixture of caged-biotin-BSA and BSA. The confocal results are listed in Table 1. The surface coverage of an adsorbed film of BSA varies considerably, depending on the solution density of BSA, the chemical nature of the substrate, and the orientation of the adsorbed protein. For example, theoretical monolayer surface densities have been reported between 2 and 7.5 ng/mm2.20,21 Glass substrates adsorbed with solutions of either 100% BSA or 100% caged-biotin-BSA yielded surface densities of 5.4 ( 1.1 and 6.8 ( 1.3 ng/mm2, respectively. Radiolabeling produced similar surface densities of 6.6 ( 1.6 ng/mm2 for BSA and 4.8 ( 0.6 ng/ mm2 for caged-biotin-BSA. The confocal measurements also reveal that surfaces preadsorbed with a 3:1 mixture of caged-biotin-BSA and BSA receive at least a monolayer of protein, more than half of which is caged-biotin-BSA at a surface density of 4.4 ( 1.4 ng/mm2. Owing to the standard deviations, however, there is no statistical difference between these five surface densities (p > 0.05). Molar binding efficiencies can be estimated from the values listed in Table 1. Streptavidin binding to irradiated mixed monolayers of caged-biotin-BSA/BSA has a molar efficiency (i.e., per molecule) on the order of 23%, while the binding of biotinylated BSA to patterned streptavidin is about 17% efficient. Combined, this yields and overall system efficiency of no more than 4%. Similarly, the ratio of protein surface densities in the irradiated and unirradiated regions were 6.25 and 4.0, respectively, for streptavidin and biotinylated BSA. Again, these ratios (20) Go¨lander, C. G.; Kiss, E. J. Colloid Interface Sci. 1988, 121, 240-253. (21) Brynda, E.; Cepalova, M. A.; Stoˆl, M. J. Biomed. Mater. Res. 1984, 18, 685-693.
Figure 3. Raw images of USAF test target (Melles Griot) patterns on caged-biotin-BSA developed with streptavidinFITC; (a-c) 1-mm-thick glass slide substrate, with a 4× magnification; (d-f) ∼0.15-mm thick glass coverslip, (d) at 4× and (e,f) at 10× magnification; and (g) 25% scaled image of a 1-mm pattern on a 1-mm-thick glass slide at 4× magnification.
are somewhat uncertain because the protein surface densities measured in the unirradiated regions are not statistically different from zero (p > 0.05). Interestingly, fluorescent streptavidin incubated with BSA and BSAbiotin-MeNPOC (no irradiation) surfaces resulted in the same low binding densities. By comparison, there is some increase in streptavidin binding in the masked regions after irradiation which can be attributed either to partial deprotection or to removal of BSA from the surface during processing. Pattern Resolution. The feature resolution of our system was examined by patterning FITC-streptavidin to a mixed monolayer of caged-biotin-BSA/BSA using a negative test mask with equal feature widths and interfeature spacings ranging from 2.19 µm to 1 mm. Confocal images of some representative patterns of various feature sizes on 1-mm-thick glass slides (Figure 3a-c) and 0.15mm thick glass cover slips (Figure 3d-f) are shown in Figure 3. Figure 3g is a 4× magnified image of a streptavidin feature patterned using a 1-mm slit mask. In all cases, the patterned features are broader than the dimensions specified by the mask. For example, element
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using the 1-mm slit mask. The pairs of features were patterned with interfeature spacing of 0.5, 1, and 3 mm. Each feature possesses a Gaussian-like profile with tails that extend beyond the dimensions prescribed by the mask (dashed lines). Although these features visually appear to have a width of 1.2 mm (see Figure 3g), as described above, the feature edges actually decay gradually. Note that as the features are moved closer together, the decaying edges overlap and fill the interfeature space, causing the features to merge. To characterize this edge phenomenon, the decaying edges of six separate 1-mm profiles were fit to an exponential decay
P(x) ) Pmax exp(-x/χ) + C
Figure 4. Ratio of feature spacing (s) to feature width (w) for 1-mm-thick (squares) and 0.15-mm-thick (circles) glass substrates. Solid lines are best fits of the data to eq 1.
6 in Figure 3c shows features patterned from a section of the test mask with equal 140-µm feature widths and spacing, but these features are observed to have 200-µm widths and 60-µm spacings. This produces a merging of features that becomes more pronounced with decreasing feature size. For the 1-mm-thick glass slide, features patterned from elements smaller than 70 µm (Figure 3c) were hardly discernible. For the 0.15-mm coverslip, features smaller than 17.5 µm (Figure 3f) were visually irresolvable. Figure 4 shows the ratio of the observed streptavidin feature spacing (s) to the observed feature width (w), plotted as a function of the test mask dimensions (x) from which the feature was patterned (see inset of Figure 4). The circles represent the ratios calculated for features patterned onto the 0.15-mm coverslip, and the squares are the ratios calculated for features patterned onto the 1-mm slide. In both cases the ratio of s/w decreases steadily with decreasing test pattern dimension x, with the decrease being much more pronounced for the thinner glass substrate. These result suggests that the spreading of the patterned streptavidin features is caused by the spreading of the collimated masked illumination as it transmits through the substrate during the deprotection step. A simple geometric representation of this spreading is as follows
x - 2t(tan θ) s ) w x + 2t(tan θ)
(1)
where t is substrate thickness, and θ is an angular spread of the masked illumination (see inset of Figure 4). The solid lines in Figure 4 are best fits of eq 1 to the data obtained for several feature sizes of the average angular spread of the masked illumination. The average angular spread was 2.20° ( 0.40° for the glass slide and 1.56° ( 0.21° for the glass coverslip. Applying the same analysis to the 1-mm slit mask feature (Figure 3g), with an observed feature width of 1.2 mm, suggests a 5.7° spread in the masked illumination. Pattern Profile. Figure 5 shows the profile of three pairs of adjacent FITC-streptavidin features patterned
(2)
where P is the protein concentration at a given distance from the pattern edge, Pmax is the maximum protein concentration estimated by the fit, x is the distance from the image edge in millimeters, χ is the characteristic distance of the decay (i.e., χ ) Pmax/e), and C is a threshold protein concentration, presumably due to nonspecific binding. The fitting parameters for this exponential are as follows: Pmax, 1.04 ( 0.2 (ng/mm2); C, 0.06 ( 0.01 (ng/ mm2); and χ, 0.22 ( 0.04 (mm) (R2 ) 0.977 ( 0.02). According to eq 2, the protein density has decayed to 50% of its maximum value at distance x ) 0.22 ln(0.5) ) 0.15 mm away from the intended 1-mm width of the feature, or the average full width at half-maximum of these six features is 1.3 mm, which is slightly greater than the visually observed 1.2-mm width of the 1-mm slit mask features. According to eq 2, an observed feature width of 1.2 mm suggests a visual cutoff of the decaying edge at approximately 70% of the feature maximum. Step-and-Repeat Patterning of Streptavidin. For caged-biotin-BSA to be applied reliably to generic biotinylated protein patterning it must be capable of reproducible step-and-repeat patterns of streptavidin with uniform binding densities. Using sequential irradiation and streptavidin incubation steps, a series of three 1-mm streptavidin features were patterned onto a glass slide. The surface was rinsed after each irradiation and incubated with FITC-streptavidin according to the patterning protocol. Each subsequent feature was patterned at least 3 mm from the previous feature to eliminate feature overlap. The substrate surface was divided into seven lanes labeled from right to left as A-G (see Figure 6). Irradiation proceeded from right to left, where B, D, and F are the sequentially irradiated regions and A, C, E, and G are the intervening unirradiated regions. The diminishing background from right to left results from the decay of the light coupled into the slide for waveguide imaging. The average, background-corrected, surface densities determined for three trials are listed in Table 2. Although the differences between bound protein densities of stepand-repeat lines were not statistically significant (p < 0.05), the amount of protein immobilized appeared to decrease slightly with each subsequent patterning step. The decreasing trend may be attributed to minor desorption of caged-biotin-BSA over time. With the exception of position A, about 15% nonspecific binding of FITCstreptavidin was observed in the unirradiated regions. Step-and-Repeat Patterning of Biotinylated Protein. Next, we demonstrate that a biotinylated protein binds primarily within the region specified during the step-and-repeat process. Figure 6 shows the results of three trials where three separate lines of biotinylated BSA were patterned sequentially from right to left. In each
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Figure 5. Averaged image profiles for three pairs of adjacent FITC-streptavidin features patterned using the 1-mm slit mask. Note that overlap of the decaying feature edges causes the features to merge as the spaces close together.
Figure 6. Surface plots of three trials of step-and-repeat patterning of one stripe of FITC-biotin-BSA and two stripes of biotin-BSA using the 1-mm slit mask. A-G refer to positions on the image. In each trial, the specified location of the FITCbiotin-BSA feature was shifted from position B (trial 1) to position D (trial 2) to position F (trial 3). The raw images were smoothed with two 3 × 3 spatial convolutions to reduce noise for the surface plot.
trial, one position was specified for patterning with FITClabeled biotinylated BSA, while the other two lines were patterned with unlabeled biotinylated BSA. In trial 1, position B was deprotected and incubated with streptavidin and then with FITC-biotin-BSA, while position D and then position F were deprotected and incubated with streptavidin and then with unlabeled biotin-BSA. In trial 2, the FITC-biotin-BSA was bound only in position D, with unlabeled biotin-BSA bound positions B and F. In trial 3, the FITC-biotin-BSA was bound only in position F, with unlabeled biotin-BSA bound in positions B and D. Positions A, C, E, and G are the unirradiated regions. Each trial was repeated three times. The background-corrected, averaged surface densities of FITC-biotin-BSA for the three sets of three trials are listed in Table 3. The amount of FITC-biotin-BSA
detected in the specified irradiated region of each of the three trials was not significantly different (position B of trial 1, position D of trial 2, and position F of trial 3 were not significantly different); however, the amount of FITCbiotin-BSA detected in the specified irradiated region of a given trial was significantly greater than the amount detected in all other regions (position B of trial 1 is significantly higher than positions A, C, D, E, F, and G of trial 1, and so on for positions D and F in trials 2 and 3). Only in trial 1 was the amount of FITC-biotin-BSA detected in the nonspecified positions (A, C, D, E, F, and G) significantly different from zero. This may be due to a possible artifact created because the first lane in each case was closest to the inlet port on the flow cell. Subsequent processing after patterned FITC-labeled biotin-BSA may have resulted in the flushing off of the labeled protein to the unlabeled regions. However, increased binding is not seen for position F for trial 2, which would be “downstream” from the labeled position D. Finally, three different polyclonal biotinylated fluorescently labeled goat antibodies [antimouse, antihuman, and antirabbit] were patterned using an identical stepand-repeat scheme. Figure 7 shows the resulting image profile, averaged in the y-direction. Compared to the biotinylated BSA patterns, the antibody patterns showed less contrast between the background and pattered regions, with overall lower binding densities 0.07 ( 0.03 ng/mm2 in the patterned regions, and 0.03 ( 0.01 ng/mm2 in the background regions. The lower binding densities, about half that observed for biotinylated BSA, may be attributed to the larger size of the antibody molecule, which is about 3-fold larger. Discussion The benefits of protein patterning via photolithographic deprotection have been reviewed along with other protein patterning methodologies.10 The major attraction of photodeprotection is the potential for site-specific patterning of multiple proteins using a step-and-repeat process.22 The sequential patterning of two spatially segregated antibody populations using photoprotected, i.e., caged biotin, and the subsequent two-component immunoassay, have been demonstrated.14 In brief, the patterning of biotinylated antibody involves coating glass substrates with caged biotin, followed by a three-step patterning process: (1) the selective deprotection of the caged biotin through a photomask, (2) the patterning of streptavidin onto the deprotected regions, and (3) the binding of biotinylated antibody to the patterned strepta(22) Pirrung, M.; Read, J. L.; Fodor, S. P.; Stryer, L. Large scale solid-phase synthesis of polypeptides and receptor binding screening thereof. U.S. Patent: 5,142,854.
Step-and-Repeat Protein Patterning
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Table 2. Surface Densities of FITC-Streptavidin in Step-and-Repeat Patterning surface density of FITC streptavidin (ng/mm2)a A irradiated regions unirradiated regions
0.33 ( 0.13b
B
C
1.08 ( 0.07b
0.16 ( 0.08b,c
D 1.06 ( 0.07b
E
F 0.99 ( 0.10b
0.16 ( 0.08b,c
G 0.15 ( 0.12b,c
A-G refer to positions indicated in Figure 6. Not statistically different from each other (p > 0.05). Not statistically different from zero (p > 0.05). b
a
c
Table 3. Surface Densities of FITC-Biotin-BSA in Step-and-Repeat Patterning surface density of FITC-biotin-BSA (ng/mm2)a B
D
F
A, C, E, G (average)
trial 1 0.21 ( 0.04b 0.11 ( 0.02c 0.09 ( 0.03c 0.05 ( 0.01 trial 2 0.02 ( 0.01c,d 0.17 ( 0.04b 0.01 ( 0.01c,d 0.02 ( 0.02d trial 3 0.01 ( 0.01c,d 0.02 ( 0.02c,d 0.17 ( 0.03b 0.01 ( 0.01d a A-G refer to positions in Figure 7. b Specified position for patterning of FTIC-biotin-BSA, all not statistically different from each other (p > 0.05). c Not statistically different from each other (p > 0.05). d Not statistically different from zero (p > 0.05).
Figure 7. Average profile plot for three biotinylated fluorescently labeled proteins. The lines were patterned from right to left: (1) goat antimouse, (2) goat antihuman, and (3) goat antirabbit.
vidin. A second antibody is patterned by translation of the photomask, and then steps 1-3 are repeated. The caged-biotin-BSA approach described by Pirrung and Huang15 offers two potential advantages for protein patterning: (1) the to-be-patterned substrates are readily coated with caged biotin by simply adsorbing a film of BSA labeled with caged biotin moieties, and (2) the presence of the BSA carrier molecule should reduce the nonspecific binding of streptavidin and biotinylated antibody in the unirradiated regions. The purpose of this study was to characterize the protein patterns produced by the photodeprotection of cagedbiotin-BSA. Studies were conducted to establish a patterning protocol that bound the highest density of streptavidin while maintaining the best pattern-tobackground contrast. Consistent with previous observations,23 the highest density of caged-biotin-BSA and the highest labeling of BSA with caged biotin did not strictly translate into higher surface densities of bound protein. The best results were obtained by preadsorbing glass substrates with a 3:1 molar mixture of caged-biotin-BSA (23) Zhao, S.; Reichert, W. Langmuir 1992, 8, 2785-2791.
to unlabeled BSA, where each caged-biotin-BSA was labeled with an average of 10 MeNPOC biotins. Using this formulation, the binding of FITC-streptavidin and biotinylated protein (FITC-biotin-BSA) to photodeprotected caged-biotin-BSA was examined. Calibrated confocal microscopy was used to determine the surface densities of protein bound to irradiated and unirradiated caged-biotin-BSA. From Table 1, a glass specimen preadsorbed with a 3:1 mixture of caged-biotinBSA/BSA would have a surface density of 3.9 × 1010 molecules/mm2 of caged-biotin-BSA. Using this value and the molecular densities in Table 1, both the binding of streptavidin to irradiated caged-biotin-BSA and the binding of biotinylated BSA to patterned streptavidin have molar efficiencies on the order of 20% for each step, for a combined molar efficiency (i.e., per molecule) of just 4%. It is interesting to note that the fluorescent images of the BSA, the caged-biotin-BSA, and the BSA/caged-biotinBSA mixtures all had somewhat mottled appearances, suggesting that the protein was not in a uniform coating on the surface. Characterization of feature resolution and shape showed considerable deviation from the desired steep-wall vertical profile. A test mask, with equal feature widths and spacing, was used to characterize the resolution of streptavidin films patterned onto a 1-mm thick glass slide and a 0.15-mm-thick glass coverslip. As the feature size decreases, the ratio of observed feature width to observed image spacing decreases, causing the features to merge (Figure 4). The visual resolution limits for the glass slide and coverslip were nominally 70 µm and 17.5 µm, respectively. Streptavidin features patterned using a 1-mm slit mask did not produce square-wave-shaped image protein density profiles; rather, image cross sections had Gaussian-like profiles with decaying edges that extended beyond the mask boundaries (Figure 5). Combined, these results suggest that the spreading of patterned features seen with the test mask is caused by decay of the pattern edge, which results in a filling of the interfeature space. Eventually, as patterned features spread closer and closer together, they become so overlapped that they merge. There are two interrelated explanations for this: photodegradation of the pattern due to irradiation overexposure and spreading of the masked illumination beyond the boundaries defined by the photomask. In IC technology, overexposure of photoresists results in loss of patterned feature definition. A quantity often used to determine the sharpness of the transition from underexposure to overexposure is the contrast, γ:24
γ)
1 log10(Ef/Ei)
(3)
where for negative photoresists, Ei is the irradiation dose at which the photoresist starts becoming insoluble, and Ef is the irradiation dose at which all of the photoresist (24) Runyan, W. R.; Bean, K. E. Semiconductor integrated circuit processing technology; Addison-Wesley: Reading, MA, 1990.
4250 Langmuir, Vol. 14, No. 15, 1998
is completely insoluble. The reverse applies to positive photoresists. In either case, the smaller the contrast, the greater the energy difference between initiating and completing development. Negative photoresists work analogously to protein patterning by photodeprotection, i.e., the irradiated region produces the feature of interest after development. Good contrasts for negative resists are on the order of 5-6, indicating a that a narrow range of irradiation doses is desirable for developing the photoresist. (Robert Hardman, Microlithography Chemical Company, private communication.) Similar definitions can be generated for our protein patterning system, where Ei is the irradiation dose at which protein patterning would be initiated, and Ef would be the irradiation dose beyond which no additional protein binding within the patterned region is observed. Referring to Figure 2, Ei and Ef indicate the irradiation doses at which the bound amount is 10% and 90% of the apparent maximum streptavidin value, respectively. Plugging Ei ) 0.5 min and Ef ) 7 min into eq 3 yields a contrast of 0.9. (Note: 1 min of irradiation at 7 mW/cm2 ) 420 mJ/cm2.) Although considerably different from photoresist, this result suggests that the caged-biotin-BSA film is sensitive to a broad range of dosages below the 8 min irradiation time used in this study (i.e., below 3.4 J/cm2) that are capable of causing at least partial deprotection. The most likely source of image broadening beyond the photomask borders is the spreading of the masked illumination between the photomask and the adsorbed film of caged-biotin-BSA. In back masking, the mask is placed on the side of the substrate opposite to that which contains the to-be-irradiated film. For collimated incident radiation, the light beam passing through the mask can be broadened by divergence of the incident irradiation, by diffraction at the slit, by scatter as the light passes through the substrate, and by reflections off of the various interfaces and surfaces encountered by the transmitted light. Divergence of the illuminating irradiation at the mask is unlikely because our source is both collimated and placed 15 cm above the irradiated specimen. In addition, the spreading of our patterned features far exceeds the diffraction limit. This implicates scatter and spurious reflections caused by the glass substrate. The effect of substrate scatter on feature broadening is depicted in Figure 4. Given the apparent susceptibility of the MeNPOC system to deprotection at irradiation doses of less than 3.4 J/s, the system would be very sensitive to feature broadening caused by scatter and reflection of the masked illumination. Improvement in resolution from 70 µm to 17.5 µm is seen by reducing the thickness of the glass substrate from 1 mm to 0.15 mm. Thus, a 7-fold reduction in substrate thickness produced a 4-fold im-
Blawas et al.
provement in resolution the scattering path length of the masked illumination. Despite feature broadening, the caged-biotin-BSA system yields reproducible step-and-repeat patterning of proteins with modest levels of nonspecific binding in the unirradiated regions. We were also able to demonstrate the patterning of three different biotinylated antibodies using this method. However, there are at least four aspects of this study that were somewhat troubling. First, is that the efficiency of each step of the process was on the order of 20%, resulting in a low overall system efficiency of 4%. One possible source of this low efficiency is that the BSA carrier of the caged-biotin-BSA intended to reduce nonspecific binding in the unirradiated regions may also inhibit the specific binding of streptavidin in the irradiated region. Alternatively, the irradiation process itself could degrade a significant fraction of the BSA-linked biotin moieties, thus reducing the density of streptavidin binding sites. Evidence for irradiation-induced biotin degradation comes from the observation that irradiation times of greater than 20 min reduced the streptavidin binding capacity of the adsorbed caged-biotin-BSA films. A second concern is the potential inaccuracy of our calibration technique, which consisted of using adsorbed, dried films of fluorescently labeled protein to construct curves of protein surface density versus fluorescence intensity. Although our patterned films were themselves assessed in the dried state under the same conditions, it is possible that making these measurements in the hydrated state would have been a better, albeit more complicated approach. A third concern is that some of our patterns appeared mottled, indicating nonuniform coverage of the patterned protein film. However, this nonuniformity was most evident on the glass coverslips, suggesting that the adsorption of the caged-biotin-BSA was sensitive to the type of substrate used. Finally, the biotin-MeNPOC ester used to tether caged-biotin to BSA was prone to moisture degradation, and its shelf life was minimal. Therefore, fresh lots of the ester needed to be made continuously. Acknowledgment. Support from NIH Grant HL 32132 and from the NSF sponsored Duke/North Carolina Center for Emerging Cardiovascular Technologies (A.S.B.) is gratefully acknowledged. Carol Fierke, Hisham Massoud, and Ashutosh Chilkoti of Duke University, James Herron of the University of Utah, and Robert Hardman of Microlithography Chemical Company are thanked for many insightful discussions. We also thank anonymous reviewers for several helpful suggestions. LA971231V
